NO146542B - STABILIZED DRY CULTURES OF Lactic Acid-Producing Bacteria - Google Patents

Description

This invention relates to stabilized, dried culture medium solids which constitute a concentrate of viable; harmless lactic acid-producing bacterial cells.

Cultures of lactic acid bacteria for the commercial production of cheese are usually preserved by freezing, using liquid nitrogen, the culture, which must therefore necessarily be stored and distributed in a frozen state.

Such kr.yogen preservation is reasonably satisfactory when the manufacturer of the culture and the cheese manufacturer have the necessary freezing and cooling equipment. Frozen cultures of certain lactic acid bacteria are also used for the commercial production of yoghurt and soured milk products.<v> For home production of yoghurt and soured milk products, frozen cultures are not satisfactory, which is due to their relatively high price and problems associated with their handling.

In Europe and the USA it has therefore been commercial practice to distribute dried cultures of yoghurt and sour milk producing bacteria for home use. With such dried cultures, serious problems have occurred with regard to their shelf life. Although retailers have often taken care to keep the dried cultures under refrigeration, the products have an undesirably short shelf life. The number of viable bacteria continuously decreases with time during refrigerated storage.

Without storage under refrigeration conditions, the dried cultures are quickly deactivated. In order to ensure that there will be sufficient viable cells present to form yoghurt and sour milk products according to the manufacturer's instructions, it has been necessary to deliver a 50 - 200% excess of the cells. This increases the costs and makes the instructions for use that follow the products inaccurate.

Similar problems arise in the manufacture and distribution of yoghurt and sour milk bacteria in the form of tablets for oral consumption. The tableted, dry cells can be stored under refrigeration, but even then there is a loss of cell viability over time. When the tablets on the packaging are stated to contain a certain minimum amount of active cells per tablet, the manufacturer must therefore, to be on the safe side, introduce a surplus of cells at the time when the tablets are manufactured to ensure that the labeling will remain valid and accurate while the product is in stock at the retailer.

Until the present invention, it was known that commercially dried cultures of lactic acid-producing bacteria do not have sufficient stability for them to be stored at room temperature. A sufficient shelf life could not be achieved unless the cultures were stored at refrigerated temperatures. The present method makes it possible for the first time to distribute and retail dried yogurt and sour milk cultures for home use and tableted yogurt and sour garlic bacteria without the need for any refrigeration.

According to the invention, there are thus provided stabilized, dried culture medium solids which constitute a concentrate of viable, harmless lactic acid-producing bacterial cells, which solids are produced by drying a fermented culture containing these cells together with other solids that are present at the end of the incubation of a culture of the cells in an aqueous nutrient medium, which medium has been dried to a moisture content of less than 5% by weight, after its pH value has been adjusted to a value favorable to the stability of the cells after drying. The new stabilized, dried culture medium substances are characteristic in that there is present in the solids a synergistically acting combination of stabilization potentiators comprising (a) an ascorbate compound selected from L-ascorbic acid and edible, water-soluble salts of this acid, and (b) a second potentiator selected from glutamic acid, aspartic acid and edible, water-soluble salts of these acids, which potentiators have been dissolved in the fermented culture to achieve effective contact with the cells before drying and in amounts corresponding, on a molar basis, to 4-20 parts by weight of L-ascorbic acid in the case of the ascorbate compound , and for the second potentiator corresponding to 1.5 - 20 parts by weight, preferably 3-15 parts by weight, of monosodium glutamate per 100 parts by weight of the total anhydrous weight of the cells and the other fermentation solids is .

The term "lactic acid bacteria" is used here to denote the large class of harmless lactic acid-producing bacteria. These bacteria generally have an ability to ferment simple carbohydrates, e.g. lactose or fructose, lactic acid being at least one of the fermentation products, and usually the one present in the greatest quantity. Examples of such lactic acid bacteria include the following: Streptococcus lactis, Streptococcus cremoris, Streptococcus diacetilactis, Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus bifidus, Lactobacillus casei, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus delbrueckii, Lactobacillus thermophilus, Lactobacillus fermentii and Pediococcus cerevisiae.

The present invention is particularly useful and offers particular advantages in connection with the production of stabilized dried bacterial concentrates of Streptococcus thermophilus, Lactobacillus bulgaricus and Lactobacillus acidophilus, but the invention is not limited to the production of dried bacterial concentrates of these three organisms. The bacteria Streptococcus thermophilus and Lactobacillus bulgaricus are sometimes referred to

the "yoghurt bacteria" and are generally used for the production of yoghurt. In certain countries, Streptococcus thermophilus and Lactobacillus acidophilus are used to produce sour milk products. In the United States, a "yoqhurt" culture is most often a mixture of Streptococcus thermophilus and Lactobacillus bulgaricus. In connection with the present invention is used

however, the term "yoghurt bacteria" generally and includes one or more strains of Lactobacillus bulgaricus, Streptococcus thermophilus or Lactobacillus acidophilus, or a combination of two or three of these species.

In the production of the stabilized, dried culture medium solids according to the present invention, a pure or mixed culture of the desired lactic acid-producing bacteria is grown in a liquid medium which provides sufficient growth of

the relevant culture(s). This medium can be composed of protein or protein fractions, various fermentable carbohydrates, growth stimulants, inorganic salts, buffers, etc., or the medium can be sterile sweet milk, skimmed milk, whey or other natural substrates, or they can be combinations thereof . The growth medium can be heated or sterilized to potentiate satisfactory growth. After the desired heat treatment, cooling and inoculation, the culture is allowed to develop under generally optimal incubation conditions with respect to time and temperature. Depending on the organism(s) being cultivated, the incubation times can be 4-48 hours, and the temperatures can vary from 15 to 50° C. It may also be desirable to regulate the pH value and the amount of dissolved oxygen.

When satisfactory growth has been achieved, the culture is cooled

in its growth medium to between 0° and 15° C.

In general, the method used to obtain viable cells of lactic acid-producing bacteria does not in itself include previously unknown steps, but it is carried out in accordance with known methods for cultivating such bacteria. When a satisfactory bacterial population has been achieved in a suitable growth medium, the pH value of the growth liquid may be lower than that which is desirable for the production of a dried product. The final pH value will typically be in the range 4.4 - 5.4. Before drying the fermentation liquid, it is advantageous to add an alkaline reagent, e.g. sodium hydroxide, to raise the pH to a level that is more favorable for the stability of the bacteria. In general, as is well known, it is desirable to regulate the pH value upwards towards neutrality (pH value 7), and regulation is carried out to at least a pH value of 5.8. Any alkali acceptable in foodstuffs can be used, e.g. sodium hydroxide, potassium hydroxide, ammonium hydroxide and calcium hydroxide. It is preferable to set the pH value to approx. 6.0 - 6.5. As a specific example, the pH value can be raised to approx. 6.2. When other additives are to be incorporated into the growth medium, which will affect the growth medium's pH value, e.g. the stability enhancers used according to the present invention, the pH regulation can conveniently be carried out at the end.

Before drying the cells in the growth medium, a combination of potentiators is added. An ascorbate compound is one of the essential contributory potentiators to achieve the increased stability. The term "ascorbate compound" shall here denote L-ascorbic acid (vitamin C) and its water-soluble edible salts. Such "edible salts" are the salts that are approved for use in human food and are of food quality. The ascorbate compound should be incorporated in an amount which, on a mole basis, corresponds to 4-20 parts by weight of L-ascorbic acid per 100 parts dry matter in the growth medium, which means the dry weight of the bacterial cells + the dry weight of the other solids found in the growth medium. On the same basis, the second potentiator is added in an amount which, on a molar basis, corresponds to 1.5 - 20 (or preferably 3-15) parts of monosodium glutamate per 100 parts of the aforementioned total dry matter content in the fermentation medium. The terms "glutamate compound" and "aspartate compound" shall be used here to refer respectively to (a) glutamic acid and its edible water-soluble salts, and (b) aspartic acid and its edible water-soluble salts. A glutamate compound is preferred which can conveniently be added in the form of monosodium glutamate.

When the growth medium is dried by freeze drying, it is desirable to incorporate a freeze protection agent. Suitable known antifreeze agents are e.g. inositol, sorbitol, mannitol, glucose, sucrose, corn syrup, dimethylsulfoxide, starches and modified starches of all types, polyvinylpyrrolidone, maltose or other mono- and disaccharides. The addition amount can be in the range from 1.0 to 300 g/l of the culture, depending on the identity of the medium in question.

An amount should be used that effectively reduces cell damage by freezing to a minimum. When another drying method is used, e.g. heat drying, no freeze protection agent is used, and generally any of the various methods for drying bacteria or useful biological materials into a powder can be used. Examples of these include freeze drying, spray drying, roller and/or vacuum pan drying. The preferred drying methods for realizing the invention are freeze drying or spray drying.

Comparison experiments that show the important and the unexpected

, results obtained by the present invention, appear from the following experiments A, B, C and D and the associated tables.

Try A

Two 2000 ml samples of reconstituted skimmed milk, (12% solids) are inoculated with an active milk subculture of Lactobacillus acidophilus in an amount of 1.0% and incubated for 8 hours at 40°C. Both samples are then cooled to 7° C. Both samples' pH value is below 5.0 after incubation and cooling. 60 minutes before freeze-drying, one 2000 ml sample is adjusted to pH 6.55 with 50% sodium hydroxide solution, and the other sample (the control sample) must still have a pH of 4.77. Both samples are then freeze-dried. The shelf life of the dried powders obtained at 21° C is shown in table A below. The analysis to determine the number of viable organisms was carried out according to a group plate counting method for determining viable organisms using a standard yogurt agar as plate culture medium. The plates are incubated at 37° C for 72 hours.

Attempt B

The conditions and methods are the same as in experiment A

with the difference that only one sample of 2000 ml is produced. 25 g of monosodium glutamate (MSG) is added to this, and the pH value is adjusted to 6.55 with a 50% sodium hydroxide solution. After a waiting time of 60 minutes for equilibrium setting, the fermentation medium containing the Lactobacillus acidophilus cells is freeze-dried. The shelf life is determined at 21° C and the following results are obtained:

Attempt C

Similar conditions are used as described in experiment A, but this time both 200 ml samples are adjusted to pH 6.00 before freeze-drying. To the one 2000 ml sample, 25 g of L-ascorbic acid (which is mixed before the pH adjustment) is also added. A pH of 6.00 is used instead of 6.55 because of the lower resulting moisture content in the powder. Both powders are freeze-dried. The shelf life of the dried powders obtained at 21° C is shown in table C below.

Try D

2000 ml of Lactobacillus acidophilus culture is prepared as described under experiment A with the difference that the pH value as in experiment C is set to 6.00 with 50% sodium hydroxide

solution after adding 25 g of L-ascorbic acid and 25 g of monosodium glutamate (MSG). The sample is freeze-dried. The results of the storage shelf life survey appear in table D.

The above test results can be most easily compared on the basis of the following summary table.

Summary table

(Attempt A, B, C and D)

Embodiments of the present invention, which can be adapted to commercial operation, are listed in examples 1 - 4 below. It will be understood that the negative controls labeled "old method" are only included for comparison purposes, while the treatments labeled "according to the invention " are embodiments of the present invention.

Example 1

Two 2000 ml samples of reconstituted skimmed milk (12% skimmed milk solids) are heated for 12 minutes at 116° C. They are then cooled to 40° C and inoculated with 1.0% of a suitable culture of Lactobacillus bulgaricus containing approx. 1 billion viable cells per ml. The two samples inoculated in this way are then incubated at 40° C for 8 hours, after which they are rapidly cooled in ice water to 5° C. The one fermented culture of 2000 ml is designated "according to the invention" and receives the following treatment: 40 g of ascorbic acid, 25 g of monosodium glutamate (MSG) and 25 g of inositol are added with constant stirring, and then the pH is adjusted to 6.10 with a 50% sodium hydroxide solution. The other 2000 ml fermented culture, which is termed "old method", does not undergo this treatment. The latter culture is control. The two cultures are then freeze-dried in a conventional manner, and the powders obtained are stored at 21° C, and appropriate plate counts for determining the number of viable organisms are carried out on Hansen's yoghurt agar, partly immediately and partly after 1, 2 and 3 the course of months. The results appear in Table I below:

Example 2

The same method as described in example 1 is used, with the change that Lactobacillus helveticus is used as the test organism. The shelf life tests with the obtained freeze-dried powders give the results listed in Table II below:

Example 3

The method described in example 1 is used with the following difference: Two 10,000 ml samples of reconstituted skimmed milk (16% solids) are used as culture media; the test organism is Lactobacillus acidophilus; the two obtained fermented cultures are spray-dried on a small spray-drying apparatus with a supply rate for the culture of 12 - 15 ml/min, a nozzle speed of 3.5 kg/cm^ and an outlet temperature of 50 - 60° C.

The shelf life results achieved with these two types of spray-dried powders are shown in the table below

III.

Example 4

The method described in example 1 is used with the following difference: Streptococcus cremoris is used as the test organism. Incubate at 21° C for 16 hours; is added per 2000 ml fermented culture 20 g ascorbic acid, 12.5 g monosodium glutamate and 12.5 g inositol; The pH value is set to 6.25 with 50% sodium hydroxide solution; an untreated culture is still used as a control; the culture media are prepared as two 2000 ml samples of reconstituted skimmed milk (16% solids), and a batch of milk agar (Ellikers) is used for the plate count determination of the number of viable organisms. The results appear in table IV below.

Example 5

Conditions and methods are the same as described in example 1, with the difference that instead of MSG, 23 g of sodium aspartate is used. The results appear in table V below.

As can be seen from the above examples, before drying both potentiators are incorporated into the culture medium and the pH value is adjusted. It is convenient to make the pH setting last as the addition of the potentiators will cause a small change in the pH value. These additives should be dissolved in the liquid: medium to achieve effective contact with the cells before drying. A short waiting period after the addition of the potentiators and after the pH setting is desirable. The cells should be allowed to settle into equilibrium with the additives. The minimum holding time has not been determined, but in general it is desirable to keep the cells in contact with the dissolved additives for 30 - 60 minutes or longer. In commercial operation, a holding time of 1 - 2 hours has been used. It is not

necessary to have a longer holding time. The culture medium containing the additives is preferably kept at a temperature between 0° and 15° C, and the temperature used is a non-freezing temperature at which the cells are protected against loss of viability. When the commercial processing of a liquid culture to obtain a dry product is delayed, e.g. overnight, the holding time can be continued under the specified cooling conditions. In the presence of the additives and when stored at a non-freezing temperature of below 15° C, the cells in the culture medium remain viable after several days of storage. However, there is no reason to delay drying, and usually the culture is subjected to freeze-drying or spray-drying within a few hours (2-6 hours) of the incorporation of the additives.

The term "dried" is used here to denote products that contain no more than 5% moisture by weight. Such products appear immediately in the form of a fine powder or granules. Generally, the average moisture content of the formed product can be reduced to at least 2.5 - 3.5% by weight by either freeze drying or spray drying. There is no minimum moisture content for the dried product, but in practice it is difficult to produce products that contain less than approx. 1 - 2% by weight water. Preferably, the stabilized dried concentrates of lactic acid-producing bacterial cells, which are produced according to the invention, contain less than 3.5% by weight of water, e.g. 2.5 - 3% moisture by weight.

When it is desired to produce tablets from the stabilized dried concentrates with a high number of viable cells, the stabilized dried material can be mixed with a

tableting suitable sugar, e.g. lactose or sucrose, whichever sugar material is preferred

in granular form tailored to its function as a tablet binder. For example, 5-10 parts by weight of the stabilized dried fermentation solids containing the cell concentrate can be mixed with 90-95 parts of tableting sugar material, and the mixture can be pressed into tablets on ordinary tableting machines.

Conventional freeze-drying and spray-drying equipment can be used there for the purposes of the present invention. In the above examples, the freeze-drying is carried out with a freeze-drying machine manufactured by Alloy Products. Inc., Waukesha, Wisconsin, and the spray drying is carried out in a Nichols/Niro laboratory spray drying equipment.

Claims (4)

1. Stabilized, dried culture medium solids which constitute a concentrate of viable, harmless lactic acid-producing bacterial cells, which solids are produced by drying a fermented culture containing these cells together with other solids present at the end of the incubation of a culture of the cells in an aqueous nutrient medium, which medium has been dried to a moisture content of less than 5% by weight, after its pH value has been adjusted to a favorable value for the stability of the cells after drying, characterized in that there is present in the solids a synergistically acting combination of stabilization potentiators comprising (a) an ascorbate compound selected from L-ascorbic acid and edible, water-soluble salts of this acid, and (b) a second potentiator selected from glutamic acid , aspartic acid and edible, water-soluble salts of these acids, which potentiators have been dissolved in the fermented culture to achieve effective contact with the cells before drying and in amounts corresponding, on a molar basis, to 4-20 parts by weight of L-ascorbic acid in the case of the ascorbate compound. and for the second potentiator corresponding to 1.5 - 20 parts by weight, preferably 3-15 parts by weight, of monosodium glutamate per 100 parts by weight of the anhydrous total weight of the cells and the other fermentation solids. 2. Stabilized, dried culture medium solids according to claim 1, characterized in that the second potentiator is monosodium glutamate. 3. Stabilized, dried culture medium solids according to claim 1 or 2, characterized in that the bacterial cells are Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus or mixtures thereof. 4. Stabilized, dried culture medium solids according to claims 1 - 3r characterized in that the fermented culture has been subjected to spray drying.